RESUMO
Maximal standard-of-care (SOC) management could not stop the life-threatening progression of a necrotizing fasciitis induced by Panton-Valentine Leukocidin-producing Methicillin-Resistant Staphylococcus aureus (MRSA) in a 12-year-old boy. Multi-route phage therapy was initiated along with antibiotics against Staphylococcus aureus, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, eventually leading to full recovery with no reported adverse events.
Assuntos
Antibacterianos , Toxinas Bacterianas , Exotoxinas , Fasciite Necrosante , Leucocidinas , Staphylococcus aureus Resistente à Meticilina , Terapia por Fagos , Pseudomonas aeruginosa , Infecções Estafilocócicas , Humanos , Masculino , Criança , Exotoxinas/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Antibacterianos/uso terapêutico , Antibacterianos/administração & dosagem , Fasciite Necrosante/terapia , Fasciite Necrosante/microbiologia , Fasciite Necrosante/tratamento farmacológico , Infecções Estafilocócicas/terapia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Terapia por Fagos/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Resultado do Tratamento , Stenotrophomonas maltophilia/efeitos dos fármacosRESUMO
Alpha toxin has become the subject of research in recent years. The objective of this article was to review and summarize recent research on the molecular structure and biological function of the alpha toxin of Clostridium perfringens. This includes the work of our research team, as well as that of other researchers. Clostridium perfringens is an anaerobic, spore-forming, Gram-positive bacillus. It can cause various intestinal diseases, such as gas gangrene, food poisoning, non-foodborne diarrhea, and enteritis. Clostridium perfringens can be classified into 5 toxinotypes A, B, C, D, and E, based on the production of major toxins. Each type of C. perfringens produces alpha toxin, which is one of the most important lethal and dermonecrotic toxins and is considered a primary virulence factor. Alpha toxin is a multifunctional metalloenzyme with phospholipase C and sphingomyelinase activities that simultaneously hydrolyze phosphatidylcholine and sphingomyelin. It can therefore destroy the integrity of cell membranes and eventually cause cell lysis. The clinical effects of alpha toxins are characterized by cytotoxicity, hemolytic activity, lethality, skin necrosis, platelet aggregation, and increased vascular permeability. Future research will concentrate on the pathogenesis of a lpha toxin exposure, clarifying the interaction between alpha toxin and the cell membrane and investigating the mechanism of activating platelet function. This research will have substantial theoretical and practical value in controlling disease progression, identifying targeted therapeutic sites, and reducing the toxic effects of vaccines.
La toxine alpha est devenue l'objet de recherches ces dernières années. L'objectif de cet article était de passer en revue et de résumer les recherches récentes sur la structure moléculaire et la fonction biologique de la toxine alpha de Clostridium perfringens. Cela inclut les travaux de notre équipe de recherche, ainsi que ceux d'autres chercheurs. Clostridium perfringens est un bacille anaérobie, sporulé et à Gram positif. Il peut provoquer diverses maladies intestinales, telles que la gangrène gazeuse, une intoxication alimentaire, de la diarrhée non alimentaire et une entérite. Clostridium perfringens peut être classé en 5 toxinotypes A, B, C, D et E, en fonction de la production des principales toxines. Chaque type de C. perfringens produit de la toxine alpha, qui est l'une des toxines létales et dermonécrotiques les plus importantes et est considérée comme un facteur de virulence primaire. La toxine alpha est une métalloenzyme multifonctionnelle possédant des activités de phospholipase C et de sphingomyélinase qui hydrolysent simultanément la phosphatidylcholine et la sphingomyéline. Elle peut donc détruire l'intégrité des membranes cellulaires et éventuellement provoquer une lyse cellulaire. Les effets cliniques des toxines alpha sont caractérisés par une cytotoxicité, une activité hémolytique, une létalité, une nécrose cutanée, une agrégation plaquettaire et une augmentation de la perméabilité vasculaire. Les recherches futures se concentreront sur la pathogénèse de l'exposition à la toxine alpha, en clarifiant l'interaction entre la toxine alpha et la membrane cellulaire et en étudiant le mécanisme d'activation de la fonction plaquettaire. Ces recherches auront une valeur théorique et pratique substantielle pour contrôler la progression de la maladie, identifier les sites thérapeutiques ciblés et réduire les effets toxiques des vaccins.(Traduit par Docteur Serge Messier).
Assuntos
Toxinas Bacterianas , Clostridium perfringens , Fosfolipases Tipo C , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Clostridium perfringens/patogenicidade , Animais , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/química , Infecções por Clostridium/veterinária , Infecções por Clostridium/microbiologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genéticaRESUMO
Clostridioides difficile toxin B (TcdB) is the key virulence factor accounting for C. difficile infection-associated symptoms. Effectively neutralizing different TcdB variants with a universal solution poses a significant challenge. Here we present the de novo design and characterization of pan-specific mini-protein binders against major TcdB subtypes. Our design successfully binds to the first receptor binding interface (RBI-1) of the varied TcdB subtypes, exhibiting affinities ranging from 20 pM to 10 nM. The cryo-electron microscopy (cryo-EM) structures of the mini protein binder in complex with TcdB1 and TcdB4 are consistent with the computational design models. The engineered and evolved variants of the mini-protein binder and chondroitin sulfate proteoglycan 4 (CSPG4), another natural receptor that binds to the second RBI (RBI-2) of TcdB, better neutralize major TcdB variants both in cells and in vivo, as demonstrated by the colon-loop assay using female mice. Our findings provide valuable starting points for the development of therapeutics targeting C. difficile infections (CDI).
Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Clostridioides difficile , Microscopia Crioeletrônica , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/química , Animais , Clostridioides difficile/metabolismo , Clostridioides difficile/genética , Clostridioides difficile/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Camundongos , Feminino , Infecções por Clostridium/imunologia , Infecções por Clostridium/microbiologia , Ligação Proteica , Humanos , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Modelos MolecularesRESUMO
Background: The tcdA gene codes for an important toxin produced by Clostridioides difficile (C. difficile), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene. Methods: Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction. Results: At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X2 = 47, p < 0.01). Conclusion: LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile, and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA-LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Infecções por Clostridium , Enterotoxinas , Fezes , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Clostridioides difficile/genética , Clostridioides difficile/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Toxinas Bacterianas/genética , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/microbiologia , Fezes/microbiologia , Fezes/química , Enterotoxinas/genética , Primers do DNA/genética , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Adulto , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Characteristics of non-clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) especially from fishery environment are poorly understood. This research, in addition to comprehensive characterisation, sought to delineate the genetic relatedness between the MRSA strains originating from clinical as well as non-clinical settings. Out of 39 methicillin-resistant staphylococcal isolates from 197 fish samples, 6 (Three each of methicillin-resistant S. haemolyticus (MRSH) and MRSA) with distinct resistance profiles were selected for whole-genome sequencing. Using respective bioinformatics tools, MRSA genomes were comprehensively characterized for resistome, virulomes, molecular epidemiology and phylogenetic analysis. Simultaneously, MRSH genomes were specifically examined to characterize antimicrobial resistance genes (ARGs), owing to the fact that MRSH is often recognized as a reservoir for resistance determinants. RESULTS: Three MRSA clones identified in this study include ST672-IVd/t13599 (sequence type-SCCmec type/spa type), ST88-V/t2526, and ST672-IVa/t1309. Though, the isolates were phenotypically vancomycin-sensitive, five of the six genomes carried vancomycin resistance genes including the VanT (VanG cluster) or VanY (VanM cluster). Among the three MRSA, only one harbored the gene encoding Panton-Valentine Leukocidin (PVL) toxin, while staphylococcal enterotoxin (SEs) genes such as sea and seb, associated with staphylococcal food poisoning were identified in two other MRSA. Genomes of MRSH carried a composite of type V staphylococcal cassette chromosome mec (SCCmec) elements (5C2 & 5). This finding may be explained by the inversion and recombination events that may facilitate the integration of type V elements to the SCC elements of S. aureus with a methicillin-susceptible phenotype. Phylogenetically, MRSA from a non-clinical setting displayed a considerable relatedness to that from clinical settings. CONCLUSION: This study highlights the genetic diversity and resistance profiles of MRSA and MRSH, with non-clinical MRSA showing notable relatedness to clinical strains. Future research should explore resistance gene transfer mechanisms and environmental reservoirs to better manage MRSA spread.
Assuntos
Peixes , Genoma Bacteriano , Staphylococcus aureus Resistente à Meticilina , Filogenia , Intoxicação Alimentar Estafilocócica , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Animais , Peixes/microbiologia , Intoxicação Alimentar Estafilocócica/microbiologia , Genoma Bacteriano/genética , Antibacterianos/farmacologia , Sequenciamento Completo do Genoma , Virulência/genética , Testes de Sensibilidade Microbiana , Humanos , Fatores de Virulência/genética , Alimentos Marinhos/microbiologia , Microbiologia de Alimentos , Toxinas Bacterianas/genética , Epidemiologia Molecular , Staphylococcus haemolyticus/genética , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/isolamento & purificação , Staphylococcus haemolyticus/patogenicidadeRESUMO
Staphylococcus aureus is a notorious pathogen predominantly involved in skin and soft tissue infections, exhibiting a distinct innate sex bias. This study explores the influence of testosterone on the virulence of S. aureus and elucidates its underlying mechanisms. Utilizing a skin abscess model in intact and castrated male mice, we assessed the effects of testosterone on S. aureus pathogenicity. Compared to controls, castrated mice showed significantly reduced abscess sizes and decreased bacterial loads, highlighting the role of testosterone in modulating the severity of S. aureus infections. In vitro experiments revealed that testosterone enhances the hemolytic activity, cytotoxicity, and oxidative stress resistance of S. aureus. Real-time quantitative PCR analysis showed a significant upregulation of the genes encoding α-hemolysin (hla) and phenol-soluble modulin (psmα). Importantly, testosterone treatment significantly enhanced the expression of the accessory gene regulator (Agr) quorum-sensing system components (agrC, agrA, agrB, agrD), while the SaeRS system (saeR, saeS, and sbi) exhibited only slight changes. Gene knockout experiments revealed that deletion of agrC, rather than saeRS and agrBD, abolishes the testosterone-induced enhancement of hemolysis and gene expression, underscoring the key role of AgrC. Molecular docking simulations indicated a direct interaction between testosterone and AgrC protein, with a strong binding affinity at the active site residue SER201. This study provides new insights into the mechanistic basis of how testosterone enhances the pathogenicity of S. aureus, potentially contributing to increased male susceptibility to S. aureus infections and offering a targeted approach for therapeutic interventions.
Assuntos
Proteínas de Bactérias , Infecções Estafilocócicas , Staphylococcus aureus , Testosterona , Masculino , Testosterona/farmacologia , Testosterona/metabolismo , Animais , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Camundongos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Virulência , Infecções Estafilocócicas/microbiologia , Transativadores/genética , Transativadores/metabolismo , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum , Simulação de Acoplamento Molecular , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Abscesso/microbiologia , Hemólise , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genéticaRESUMO
The human body harbors a substantial population of bacteria, which may outnumber host cells. Thus, there are multiple interactions between both cell types. Given the common presence of Staphylococcus aureus in the human body and the role of Th17 cells in controlling this pathogen on mucous membranes, we sought to investigate the effect of α-hemolysin, which is produced by this bacterium, on differentiating Th17 cells. RNA sequencing analysis revealed that α-hemolysin influences the expression of signature genes for Th17 cells as well as genes involved in epigenetic regulation. We observed alterations in various histone marks and genome methylation levels via whole-genome bisulfite sequencing. Our findings underscore how bacterial proteins can significantly influence the transcriptome, epigenome, and phenotype of human Th17 cells, highlighting the intricate and complex nature of the interaction between immune cells and the microbiota.
Assuntos
Toxinas Bacterianas , Epigênese Genética , Proteínas Hemolisinas , Staphylococcus aureus , Células Th17 , Staphylococcus aureus/imunologia , Staphylococcus aureus/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/imunologia , Proteínas Hemolisinas/metabolismo , Humanos , Células Th17/imunologia , Células Th17/metabolismo , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/genética , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Metilação de DNA , Diferenciação Celular , TranscriptomaRESUMO
Cyanobacterial toxins are the most common algal toxins, which are highly toxic and can persist in the aquatic environment without easy degradation, posing risks to the ecosystem and human health that cannot be ignored. Although microbiological methods for the removal of cyanobacterial toxins from aqueous environments are highly efficient, their degradation efficiency is susceptible to many abiotic environmental factors. In this paper, Microcystin-LR (MC-LR) and its microbial degrading enzymes were selected to study the effects of common environmental factors (temperature (T), NO3-, NH4+, Cu2+, Zn2+) and their levels during microbial degradation of cyanobacterial toxins in aqueous environments by using molecular docking, molecular dynamics simulation, analytical factor design, and the combined toxicokinetics of TOPKAT (toxicity prediction). It was found that the addition of T, NO3- and Cu2+ to the aqueous environment promoted the microbial degradation of MC-LR, while the addition of NH4+ and Zn2+ inhibited the degradation; The level effect study showed that the microbial degradation of MC-LR was promoted by increasing levels of added T and NO3- in the aqueous environment, whereas it was inhibited and then promoted by increasing levels of NH4+, Cu2+ and Zn2+. In addition, the predicted toxicity of common Microcystins (MCs) showed that MC-LR, Microcystin-RR (MC-RR) and Microcystin-YR (MC-YR) were not carcinogenic, developmentally toxic, mutagenic or ocular irritants in humans. MC-LR and MC-RR are mild skin irritants and MC-YR is not a skin irritant. MC-YR has a higher chronic and acute toxicity in humans than MC-LR and MC-RR. Acute/chronic toxicity intensity for aquatic animals: MC-YR > MC-LR > MC-RR and for aquatic plants: MC-LR > MC-YR > MC-RR. This suggests that MC-YR also has a high environmental health risk. This paper provides theoretical support for optimizing the environmental conditions for microbial degradation of cyanobacterial toxins by studying the effects of common environmental factors and their level effects in the aquatic environment.
Assuntos
Toxinas Bacterianas , Toxinas Marinhas , Microcistinas , Microcistinas/metabolismo , Microcistinas/toxicidade , Microcistinas/química , Toxinas Marinhas/metabolismo , Toxinas Marinhas/toxicidade , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Biodegradação Ambiental , Cianobactérias/metabolismo , Toxinas de Cianobactérias , Simulação de Acoplamento Molecular , Poluentes Químicos da Água/toxicidade , Poluentes Químicos da Água/metabolismo , Simulação de Dinâmica MolecularRESUMO
The Panton-Valentine leukocidin (PVL) of Staphylococcus aureus is associated with necrotizing infections. After binding to complement 5a receptor (C5aR/CD88) and CD45 it causes cytolysis in polymorphonuclear neutrophils (PMNs) as well as inflammasome activation in monocytes. The objective of this study was to test if (ant)agonists of C5aR and CD45 can attenuate the effect of PVL on PMNs and monocytes. We tested the effect of various concentrations of six C5aR (ant)agonists (avacopan, BM213, DF2593A, JPE-1375, PMX205 and W-54011) and one CD45 antagonist (NQ301) to attenuate the cytotoxic effect of PVL on human PMNs and monocytes in vitro. Shifts in the half-maximal effective concentration (EC50) of PVL to achieve a cytotoxic effect on PMNs and modulation of inflammatory cytokine response from monocytes were determined by flow cytometry and IL-1ß detection. Pre-treatment of PMNs with avacopan, PMX205 and W-54,011 resulted in 3.6- to 4.3-fold shifts in the EC50 for PVL and were able to suppress IL-1ß secretion by human monocytes in the presence of PVL. BM213, DF2593A and NQ301 were unable to change the susceptibility of PMNs towards PVL or reduce inflammasome activation in monocytes. Avacopan, PMX205 and W-54,011 showed protection against PVL-induced cytotoxicity and suppressed IL-1ß secretion by monocytes. Clinical studies are needed to prove whether these substances can be used therapeutically as repurposed drugs.
Assuntos
Toxinas Bacterianas , Exotoxinas , Leucocidinas , Monócitos , Neutrófilos , Receptor da Anafilatoxina C5a , Staphylococcus aureus , Leucocidinas/metabolismo , Leucocidinas/antagonistas & inibidores , Exotoxinas/metabolismo , Exotoxinas/farmacologia , Exotoxinas/antagonistas & inibidores , Humanos , Toxinas Bacterianas/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/imunologia , Staphylococcus aureus/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Antibacterianos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismoRESUMO
Pantoea stewartii subsp. stewartii (Pnss), is the bacterial causal agent of Stewart's wilt of sweet corn. Disease symptoms include systemic wilting and foliar, water-soaked lesions. A Repeat-in-toxin (RTX)-like protein, RTX2, causes cell leakage and collapse in the leaf apoplast of susceptible corn varieties and is a primary mediator of water-soaked lesion formation in the P. stewartii-sweet corn pathosystem. RTX toxins comprise a large family of proteins, which are widely distributed among Gram-negative bacteria. These proteins are generally categorized as cellulolysins, but the Biofilm-Associated Proteins (Bap) subfamily of RTX toxins are implicated in surface adhesion and other biofilm behaviors. The Pnss RTX2 is most phylogenetically related to other Bap proteins suggesting that Pnss RTX2 plays a dual role in adhesion to host surfaces in addition to mediating the host cell lysis that leads to water-soaked lesion formation. Here we demonstrated that RTX2 localizes to the bacterial cell envelope and influences physiochemical properties of the bacterial cell envelope that impact bacterial cell length, cell envelope integrity and overall cellular hydrophobicity. Interestingly, the role of RTX2 as an adhesin was only evident in absence of exopolysaccharide (EPS) production suggesting that RTX2 plays a role as an adhesin early in biofilm development before EPS production is fully induced. However, deletion of rtx2 severely impacted Pnss' colonization of the xylem suggesting that the dual role of RTX2 as a cytolysin and adhesin is a mechanism that links the apoplastic water-soaked lesion phase of infection to the wilting phase of the infection in the xylem.
Assuntos
Aderência Bacteriana , Proteínas de Bactérias , Interações Hidrofóbicas e Hidrofílicas , Pantoea , Doenças das Plantas , Zea mays , Pantoea/metabolismo , Pantoea/fisiologia , Pantoea/genética , Zea mays/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Membrana Celular/metabolismo , Folhas de Planta/microbiologiaRESUMO
Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in humans and animals, induced by enterotoxins produced by these pathogens. Despite the crucial role of neutrophils in combatting bacterial infections, our understanding of how enterotoxins impact neutrophil function is limited. To address this knowledge gap, we used heat-labile enterotoxin (LT) and heat-stable enterotoxin a (STa) to investigate their impact on the effector functions of neutrophils. Our study reveals that pSTa does not exert any discernible effect on the function of neutrophils. In contrast, LT altered the migration and phagocytosis of neutrophils and induced the production of inflammatory factors via activation of cAMP/PKA and ERK1/2 signaling. LT also attenuated the release of neutrophil extracellular traps by neutrophils via the PKA signaling pathway. Our findings provide novel insights into the impact of LT on neutrophil function, shedding light on the underlying mechanisms that govern its immunoregulatory effects. This might help ETEC in subverting the immune system and establishing infection.
Assuntos
Toxinas Bacterianas , Proteínas Quinases Dependentes de AMP Cíclico , AMP Cíclico , Escherichia coli Enterotoxigênica , Enterotoxinas , Infecções por Escherichia coli , Proteínas de Escherichia coli , Neutrófilos , Fagocitose , Enterotoxinas/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Humanos , AMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/imunologia , Transdução de SinaisRESUMO
The bacterium Clostridium botulinum, well-known for producing botulinum neurotoxins, which cause the severe paralytic illness known as botulism, produces C2 toxin, a binary AB-toxin with ADP-ribosyltranferase activity. C2 toxin possesses two separate protein components, an enzymatically active A-component C2I and the binding and translocation B-component C2II. After proteolytic activation of C2II to C2IIa, the heptameric structure binds C2I and is taken up via receptor-mediated endocytosis into the target cells. Due to acidification of endosomes, the C2IIa/C2I complex undergoes conformational changes and consequently C2IIa forms a pore into the endosomal membrane and C2I can translocate into the cytoplasm, where it ADP-ribosylates G-actin, a key component of the cytoskeleton. This modification disrupts the actin cytoskeleton, resulting in the collapse of cytoskeleton and ultimately cell death. Here, we show that the serine-protease inhibitor α1-antitrypsin (α1AT) which we identified previously from a hemofiltrate library screen for PT from Bordetella pertussis is a multitoxin inhibitor. α1AT inhibits intoxication of cells with C2 toxin via inhibition of binding to cells and inhibition of enzyme activity of C2I. Moreover, diphtheria toxin and an anthrax fusion toxin are inhibited by α1AT. Since α1AT is commercially available as a drug for treatment of the α1AT deficiency, it could be repurposed for treatment of toxin-mediated diseases.
Assuntos
Toxinas Bacterianas , Toxinas Botulínicas , alfa 1-Antitripsina , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/antagonistas & inibidores , Toxinas Botulínicas/química , Humanos , alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/química , Toxinas Bacterianas/metabolismo , Toxina Diftérica/metabolismo , Corynebacterium diphtheriae/metabolismo , Corynebacterium diphtheriae/efeitos dos fármacos , Antígenos de Bactérias/metabolismo , Animais , Clostridium botulinum/metabolismo , Bacillus anthracis/metabolismo , Bacillus anthracis/efeitos dos fármacosRESUMO
Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) with Methicillin-Resistant S. aureus (MRSA) strains being a major contributor in both community and hospital settings. S. aureus relies on metabolic diversity and a large repertoire of virulence factors to cause disease. This includes α-hemolysin (Hla), an integral player in tissue damage found in various models, including SSTIs. Previously, we identified a role for the Spx adapter protein, YjbH, in the regulation of several virulence factors and as an inhibitor of pathogenesis in a sepsis model. In this study, we found that YjbH is critical for tissue damage during SSTI, and its absence leads to decreased proinflammatory chemokines and cytokines in the skin. We identified no contribution of YjbI, encoded on the same transcript as YjbH. Using a combination of reporters and quantitative hemolysis assays, we demonstrated that YjbH impacts Hla expression and activity both in vitro and in vivo. Additionally, expression of Hla from a non-native promoter reversed the tissue damage phenotype of the ΔyjbIH mutant. Lastly, we identified reduced Agr activity as the likely cause for reduced Hla production in the ΔyjbH mutant. This work continues to define the importance of YjbH in the pathogenesis of S. aureus infection as well as identify a new pathway important for Hla production.
Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Regulação Bacteriana da Expressão Gênica , Proteínas Hemolisinas , Staphylococcus aureus , Transativadores , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/imunologia , Staphylococcus aureus/genética , Camundongos , Animais , Transativadores/genética , Transativadores/metabolismo , Infecções Cutâneas Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/patologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/imunologia , Pele/microbiologia , Pele/patologia , Pele/imunologia , Fatores de Virulência/genética , Humanos , Infecções dos Tecidos Moles/microbiologia , Infecções dos Tecidos Moles/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citocinas/metabolismo , Citocinas/imunologia , Citocinas/genéticaRESUMO
The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore-forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re-examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin-luciferase reaction. Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca2+ levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca2+ mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx-sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut-like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles.
Assuntos
Trifosfato de Adenosina , Toxinas Bacterianas , Cálcio , Canais de Cloreto , Vesículas Extracelulares , Oócitos , Xenopus laevis , Animais , Oócitos/metabolismo , Oócitos/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Canais de Cloreto/metabolismo , Humanos , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Feminino , Clostridium perfringens/metabolismoRESUMO
Clostridioides difficile is a spore-forming pathogen and the most common cause of healthcare-associated diarrhea and colitis in the United States. Besides producing the main virulence factors, toxin A (TcdA) and toxin B (TcdB), many of the common clinical strains encode the C. difficile transferase (CDT) binary toxin. The role of CDT in the context of C. difficile infection (CDI) is poorly understood. Inflammation is a hallmark of CDI and multiple mechanisms of inflammasome activation have been reported for TcdA, TcdB, and the organism. Some studies have suggested that CDT contributes to this inflammation through a TLR2-dependent priming mechanism that leads to the suppression of protective eosinophils. Here, we show that CDT does not prime but instead activates the inflammasome in bone marrow-derived dendritic cells (BMDCs). In bone marrow-derived macrophages (BMDMs), the cell binding and pore-forming component of the toxin, CDTb, alone activates the inflammasome and is dependent on K+ efflux. The activation is not observed in the presence of CDTa and is not observed in BMDMs derived from Nlrp3-/- mice suggesting the involvement of the NLRP3 inflammasome. However, we did not observe evidence of CDT-dependent inflammasome priming or activation in vivo. Mice were infected with R20291 and an isogenic CRISPR/Cas9-generated R20291 ΔcdtB strain of C. difficile. While CDT contributes to increased weight loss and cecal edema at 2 days post infection, the relative levels of inflammasome-associated cytokines, IL-1ß and IL-18, in the cecum and distal colon are unchanged. We also saw CDT-dependent weightloss in Nlrp3-/- mice, suggesting that the increased weightloss associated with the presence of CDT is not a result of NLRP3-dependent inflammasome activation. This study highlights the importance of studying gene deletions in the context of otherwise fully isogenic strains and the challenge of translating toxin-specific cellular responses into a physiological context, especially when multiple toxins are acting at the same time.
Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Clostridioides difficile , Infecções por Clostridium , Inflamassomos , Inflamação , Camundongos Endogâmicos C57BL , Animais , Camundongos , Clostridioides difficile/patogenicidade , Toxinas Bacterianas/metabolismo , Inflamação/metabolismo , Inflamassomos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Infecções por Clostridium/imunologia , Infecções por Clostridium/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , ADP Ribose Transferases/metabolismo , ADP Ribose Transferases/genética , Macrófagos/metabolismo , Macrófagos/imunologia , Camundongos Knockout , EnterotoxinasRESUMO
Clostridioides difficile sequence type (ST) 35 has been found in humans and animals worldwide. However, its genomic epidemiology and clonal transmission have not been explored in detail. In this study, 176 C. difficile ST35 isolates from six countries were sequenced. Genomic diversity, clonal transmission and epidemiological data were analyzed. Sporulation and virulence capacities were measured. Four ribotypes (RT) were identified including RT046 (97.2%), RT656 (1.1%), RT427 (0.6%), and RT AI-78 (1.1%). Phylogenetic analysis of 176 ST35 genomes, along with 50 publicly available genomes, revealed two distinctive lineages without time-, region-, or source-dependent distribution. However, the distribution of antimicrobial resistance genes differed significantly between the two lineages. Nosocomial and communal transmission occurred in humans with the isolates differed by ≤ two core-genome single-nucleotide polymorphism (cgSNPs) and clonal circulation was found in pigs with the isolates differed by ≤ four cgSNPs. Notably, interspecies clonal transmission was identified among three patients with community acquired C. difficile infection and pigs with epidemiological links, differed by ≤ nine cgSNPs. Toxin B (TcdB) concentrations were significantly higher in human isolates compared to pig isolates, and ST35 isolates exhibited stronger sporulation capacities than other STs. Our study provided new genomic insights and epidemiological evidence of C. difficile ST35 intraspecies and interspecies clonal transmission, which can also be facilitated by its strong sporulation capacity.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Genoma Bacteriano , Filogenia , Ribotipagem , Clostridioides difficile/genética , Clostridioides difficile/classificação , Clostridioides difficile/isolamento & purificação , Humanos , Animais , Infecções por Clostridium/transmissão , Infecções por Clostridium/microbiologia , Infecções por Clostridium/epidemiologia , Suínos , Polimorfismo de Nucleotídeo Único , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Infecção Hospitalar/transmissão , Epidemiologia Molecular , Virulência/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Genômica , Farmacorresistência Bacteriana/genéticaRESUMO
The systemic toxicity of cyclic peptides produced by cyanobacteria (CCPs) is not yet completely understood. Apart from the most known damages to the liver and kidneys, symptoms of their neurotoxicity have also been reported. Hepatotoxic CCPs, like microcystins, as well as non-hepatotoxic anabaenopeptins and planktopeptins, all exhibit cytotoxic and cytostatic effects on mammalian cells. However, responses of different cell types to CCPs depend on their specific modes of interaction with cell membranes. This study demonstrates that non-hepatotoxic planktopeptin BL1125 and anabaenopeptins B and F, at concentrations up to 10 µM, affect normal and tumor human astrocytes (NHA and U87-GM) in vitro by their almost immediate insertion into the lipid monolayer. Like microcystin-LR (up to 1 µM), they inhibit Ser/Thr phosphatases and reorganize cytoskeletal elements, with modest effects on their gene expression. Based on the observed effects on intermediate filaments and intermediate filament linkage elements, their direct or indirect influence on tubulin cytoskeletons via post-translational modifications, we conclude that the basic mechanism of CCP toxicities is the induction of inter- and intracellular communication failure. The assessed inhibitory activity on Ser/Thr phosphatases is also crucial since the signal transduction cascades are modulated by phosphorylation/dephosphorylation processes.
Assuntos
Astrócitos , Cianobactérias , Citoesqueleto , Peptídeos Cíclicos , Humanos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Peptídeos Cíclicos/toxicidade , Citoesqueleto/efeitos dos fármacos , Toxinas de Cianobactérias , Microcistinas/toxicidade , Toxinas Marinhas/toxicidade , Toxinas Bacterianas/toxicidade , Linhagem Celular TumoralRESUMO
Cyanobacteria are cosmopolitan organisms; nonetheless, climate change and eutrophication are increasing the occurrence of cyanobacteria blooms (cyanoblooms), thereby raising the risk of cyanotoxins in water sources used for drinking, agriculture, and livestock. This study aimed to determine the presence of cyanobacteria, including toxigenic cyanobacteria and the occurrence of cyanotoxins in the El Pañe reservoir located in the high-Andean region, Arequipa, Peru, to support water quality management. The study included morphological observation of cyanobacteria, molecular determination of cyanobacteria (16S rRNA analysis), and analysis of cyanotoxins encoding genes (mcyA for microcystins, cyrJ for cylindrospermopsins, sxtl for saxitoxins, and AnaC for anatoxins). In parallel, chemical analysis using Liquid Chromatography coupled with Mass Spectrometry (LC-MS/MS) was performed to detect the presence of cyanotoxins (microcystins, cylindrospermopsin, saxitoxin, and anatoxin, among others) and quantification of Microcystin-LR. Morphological data show the presence of Dolichospermum sp., which was confirmed by molecular analysis. Microcystis sp. was also detected through 16S rRNA analysis and the presence of mcyA gene related to microcystin production was found in both cyanobacteria. Furthermore, microcystin-LR and demethylated microcystin-LR were identified by chemical analysis. The highest concentrations of microcystin-LR were 40.60 and 25.18 µg/L, in May and November 2022, respectively. Microcystins were detected in cyanobacteria biomass. In contrast, toxins in water (dissolved) were not detected. Microcystin concentrations exceeded many times the values established in Peruvian regulation and the World Health Organization (WHO) in water intended for human consumption (1 µg/L). This first comprehensive report integrates morphological, molecular, and chemical data and confirms the presence of two toxigenic cyanobacteria and the presence of microcystins in El Pañe reservoir. This work points out the need to implement continuous monitoring of cyanobacteria and cyanotoxins in the reservoir and effective water management measures to protect the human population from exposure to these contaminants.
Assuntos
Toxinas Bacterianas , Cianobactérias , Monitoramento Ambiental , Microcistinas , Peru , Cianobactérias/genética , Cianobactérias/metabolismo , Toxinas Bacterianas/análise , Toxinas Bacterianas/genética , Microcistinas/análise , Qualidade da Água , Toxinas de Cianobactérias , Microbiologia da Água , Toxinas Marinhas/análiseRESUMO
Epsilon toxin (ETX), a potential agent of biological and toxic warfare, causes the death of many ruminants and threatens human health. It is crucial to understand the toxic mechanism of such a highly lethal and rapid course toxin. In this study, we detected the effects of ETX on the proteome and phosphoproteome of MDCK cells after 10 min and 30 min. A total of 44 differentially expressed proteins (DEPs) and 588 differentially phosphorylated proteins (DPPs) were screened in the 10 min group, while 73 DEPs and 489 DPPs were screened in the 30 min group. ETX-induced proteins and phosphorylated proteins were mainly located in the nucleus, cytoplasm, and mitochondria, and their enrichment pathways were related to transcription and translation, virus infection, and intercellular junction. Meanwhile, the protein-protein interaction network screened out several hub proteins, including SRSF1/2/6/7/11, SF3B1/2, NOP14/56, ANLN, GTPBP4, THOC2, and RRP1B. Almost all of these proteins were present in the spliceosome pathway, indicating that the spliceosome pathway is involved in ETX-induced cell death. Next, we used RNAi lentiviruses and inhibitors of several key proteins to verify whether these proteins play a critical role. The results confirmed that SRSF1, SF3B2, and THOC2 were the key proteins involved in the cytotoxic effect of ETX. In addition, we found that the common upstream kinase of these key proteins was SRPK1, and a reduction in the level of SRPK1 could also reduce ETX-induced cell death. This result was consistent with the phosphorylated proteomics analysis. In summary, our study demonstrated that ETX induces phosphorylation of SRSF1, SF3B2, THOC2, and SRPK1 proteins on the spliceosome pathway, which inhibits normal splicing of mRNA and leads to cell death.
Assuntos
Toxinas Bacterianas , Fosfoproteínas , Proteoma , Animais , Cães , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Células Madin Darby de Rim Canino , Toxinas Bacterianas/toxicidade , Fosforilação , Proteômica , Mapas de Interação de Proteínas , Spliceossomos/metabolismo , Spliceossomos/efeitos dos fármacosRESUMO
The human colonic commensal enterotoxigenic Bacteroides fragilis (ETBF) is associated with chronic colitis and colon cancer. ETBF colonization induces colitis via the Bacteroides fragilis toxin (BFT). BFT secreted by ETBF cause colon inflammation via E-cadherin cleavage/NF-κB signaling. ETBF promotes colon tumorigenesis via interleukin 17A (IL-17A)/CXCL-dependent inflammation, but its bioactive therapeutics in ETBF-promoted tumorigenesis remain unexplored. In the current study, we investigated the caffeic acid phenethyl ester (CAPE) in the murine model of ETBF colitis and tumorigenesis. In this study, we observed that CAPE treatment mitigated inflammation induced by ETBF in mice. Additionally, our findings indicate that CAPE treatment offers protective effects against ETBF-enhanced colon tumorigenesis in a mouse model of colitis-associated colon cancer induced by azoxymethane (AOM) and dextran sulfate sodium. Notably, the decrease in colon tumorigenesis following CAPE administration correlates with a reduction in the expression of IL-17A and CXCL1 in the gastrointestinal tract. The molecular mechanism for CAPE-induced protection against ETBF-mediated tumorigenesis is mediated by IL-17A/CXCL1, and by NF-κB activity in intestinal epithelial cells. Our findings indicate that CAPE may serve as a preventive agent against the development of ETBF-induced colitis and colorectal cancer (CRC).